• No results found

CoIN: co-inducible nitrate expression system for secondary metabolites in Aspergillus nidulans

N/A
N/A
Protected

Academic year: 2020

Share "CoIN: co-inducible nitrate expression system for secondary metabolites in Aspergillus nidulans"

Copied!
10
0
0

Loading.... (view fulltext now)

Full text

(1)

RESEARCH

CoIN: co-inducible nitrate expression

system for secondary metabolites in

Aspergillus

nidulans

Philipp Wiemann

1,3†

, Alexandra A. Soukup

1,4†

, Jacob S. Folz

1,5

, Pin‑Mei Wang

1,6

, Andreas Noack

1

and Nancy P. Keller

1,2*

Abstract

Background: Sequencing of fungal species has demonstrated the existence of thousands of putative secondary metabolite gene clusters, the majority of them harboring a unique set of genes thought to participate in production of distinct small molecules. Despite the ready identification of key enzymes and potential cluster genes by bioinfor‑ matics techniques in sequenced genomes, the expression and identification of fungal secondary metabolites in the native host is often hampered as the genes might not be expressed under laboratory conditions and the species might not be amenable to genetic manipulation. To overcome these restrictions, we developed an inducible expres‑ sion system in the genetic model Aspergillus nidulans.

Results: We genetically engineered a strain of A. nidulans devoid of producing eight of the most abundant endog‑ enous secondary metabolites to express the sterigmatocystin Zn(II)2Cys6 transcription factor‑encoding gene aflR and its cofactor aflS under control of the nitrate inducible niiA/niaD promoter. Furthermore, we identified a subset of promoters from the sterigmatocystin gene cluster that are under nitrate‑inducible AflR/S control in our production strain in order to yield coordinated expression without the risks from reusing a single inducible promoter. As proof of concept, we used this system to produce β‑carotene from the carotenoid gene cluster of Fusarium fujikuroi.

Conclusion: Utilizing one‑step yeast recombinational cloning, we developed an inducible expression system in the genetic model A. nidulans and show that it can be successfully used to produce commercially valuable metabolites. Keywords: Yeast recombinational cloning, Secondary metabolism, Genetic engineering, Carotenes, Aspergillus nidulans, Fusarium fujikuroi, AflR, Sterigmatocystin, Nitrate, Biotechnology, Synthetic biology

© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Background

Natural products or secondary metabolites (SMs) have been invaluable as platforms for developing front-line drugs. Between 1981 and 2010, 5% of the 1031 new chemical entities approved as drugs by the Food and Drug Administration (FDA) were natural products or derivatives, including 48.6% of cancer medications [36]. In addition, SMs are major sources of innovative

therapeutic agents for both bacterial and fungal infec-tious diseases, lipid disorders, and immunomodulation [16]. Fungal SMs have proven to be a particularly impor-tant source of new leads with useful pharmaceutical activities. A literature survey of fungal metabolites, cov-ering 1500 fungal SMs that were isolated and character-ized between 1993 and 2001, showed that more than half of the molecules had antibacterial, antifungal or antitu-mor activity [41]. However, the full metabolic potential of the majority of existing fungal species has not been inves-tigated. Major roadblocks in this endeavor are that some species are not cultivable under laboratory conditions and/or their SM gene clusters are silent. Previous strate-gies on activating fungal SMs have focused mainly on (1)

Open Access

*Correspondence: npkeller@wisc.edu

Philipp Wiemann and Alexandra A. Soukup contributed equally to this work

1 Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI 53706, USA

(2)

activating endogenous gene clusters by over-expressing the pathway-specific transcription factor [13, 43, 54]), (2) manipulating global regulators [11, 28], and (3) express-ing the entire gene cluster in a heterologous host [8]. Although successful in some cases, these strategies have significant disadvantages. As not all fungal species are easily amenable to genetic manipulation, strategies that focus on endogenous activation are impossible in these species. This prevents the option of over-expressing a cluster-specific transcription factor, which has been the most successful approach to activating cryptic clusters thus far (reviewed in [51]). In addition, not all SM clus-ters contain transcription factors and although some clusters have been activated by overexpressing every gene in the cluster [14, 53], this adds labor and time to the pro-cess and may be hard to achieve with clusters containing many genes.

Previous approaches expressing fungal gene clusters in heterologous hosts (mainly Saccharomyces cerevi-siae or Aspergillus spp.) focused on amplification of the entire gene cluster including native promoters. Although these approaches lead to expression of the targeted gene clusters in some cases [55], the use of native promot-ers cannot guarantee controlled activation of the genes. Therefore, the identification and use of defined pro-moters presents an alternative means to activate clus-ters. Cloning of entire gene clusters can be achieved by PCR-based amplification of the desired DNA region and subsequent yeast recombination-based cloning [55]. Promoter exchanges using this technique rely on the identification of different promoter regions as the use of identical promoter sequences is impossible due to the homologous recombination among promoters [14].

Our goal was to identify a series of distinct promot-ers that could be activated in one step and, furthermore,

activated under an inducible system as many SMs exhibit antifungal properties that could be toxic to the heterolo-gous host [12, 46]. Thus, we designed a strain of genetic model organism Aspergillus nidulans that contains an inducible genetic construct which allows for expression of the positive acting transcriptional elements of the ster-igmatocystin (ST) gene cluster, aflR and aflS (formerly aflJ, [20]). The ST gene cluster contains 25 distinct genes and it is known that the transcription factor AflR and its cofactor AflS are responsible for ST production [9]. We constructed a strain of A. nidulans with its endogenous ST cluster removed but with aflR/aflS placed back into the strain under the control of a nitrate inducible diver-gent promoter (niiA(p)/niaD(p)) [44, 45], thereby allow-ing controlled aflR/aflS expression based on culture conditions. We tested the expression of all 25 ST pro-moters by AflR/AflS in this strain and identified eight ST promoters specifically regulated by nitrate induction of aflR/S. To test the system for expression of a fungal sec-ondary metabolite, we cloned the carotenoid gene cluster from Fusarium fujikuroi and placed it under control of these inducible ST promoters. We show that the derived A. nidulans transformants produce β-carotene in com-petitive levels to existing systems using our technology.

Methods

Fungal strains and culture conditions

Aspergillus nidulans strains used in this study are listed in Additional file 1: Table S1. Fusarium fujikuroi IMI58289 [51] was used for carRA, carB, and ggs1 ampli-fication as a reference for carotenoid production. Strains were maintained as glycerol stocks and activated on solid glucose minimal medium (GMM) at 37 °C with appropri-ate supplements [48]. For experiments in Fig. 1, nitrate was replaced with equimolar ammonium tartrate and

Fig. 1 Nitrate‑inducible aflR/S expression in A. nidulans.a Schematic overview of the aflR/S expression strain TPMW2.3 that harbors the nitrate‑ inducible niaD/niiA promoter‑driven aflR/S genes at the native sterigmatocystin cluster locus. b Northern blot analysis of nitrate‑dependent aflR/S expression in A. nidulans TPMW2.3. Strains were grown in 50 mL of GMM with 35 mM glutamine as nitrogen source supplemented with 5 mM uracil/uridine and riboflavin for 24 h at 250 rpm at 37 °C. The mycelia were washed and shifted into new media containing either NH4+ or NO3− as

(3)

supplemented with 5 mM uracil and uridine, respectively. For solidified media, Noble Agar (Difco™, BD, USA) was added at 16  g/L. For pyrG auxotrophs, the growth medium was supplemented with 5 mM uridine and ura-cil. For riboB auxotrophs, the growth medium was sup-plemented with 5 mM riboflavin. For pyroA auxotrophs, the growth medium was supplemented with 5 mM pyri-doxine. Conidia were harvested in 0.01% Tween 80 and enumerated using a hemocytometer. For RNA analy-sis, indicated strains were inoculated into 50 mL of liq-uid GMM with 35 mM glutamine as nitrogen source at 5 × 106 conidia/mL in duplicate and grown at 37 °C and

250  rpm for 24  h in ambient light conditions. The cul-tures were shifted into new GMM media either con-taining 70 mM nitrate or 35 mM glutamine as nitrogen source for 1 h. The mycelium was harvested and lyophi-lized before RNA extraction. For carotenoid production 5 × 106 conidia of indicated strains were inoculated on

20  mL liquid stationary GMM with either containing 70 mM nitrate or 35 mM glutamine as nitrogen source for 3 days at 37 °C (A. nidulans) or 29 °C (F. fujikuroi) in the dark. To distinguish between carotenoid production in the mycelia and spores, the strains were grown on liq-uid stationary GMM media (described above) for 3 days at 37 °C in the light to induce sporulation. Mycelia and spores were resuspended in 0.01% (v/v) Tween80, vor-texed to separate spores from mycelia. Spores were sepa-rated from mycelia by filtration.

Yeast recombinational cloning

Yeast strain BJ5464 (MATalpha, ura3-52, trp1, leu2-∆1, his3-∆200, pep4::HIS3, prb1-∆1.6R, can1, GAL) was inoc-ulated into 25–50 mL of appropriate media (2× YPDA) and incubated at 30 °C at 200 rpm overnight. The con-centration of overnight culture was determined using OD 600 with a 1 × 107 cells/mL set to an OD reading of

1.0. Then, 1.25 × 109 cells were centrifuged at 3000×g for

5 min. Fresh media was added to the pelleted cells and added to a baffled flask containing 250 mL of 2× YPAD to a final concentration of 5 × 106  cells/mL and

incu-bated at 30  °C at 200  rpm until the cell titer reached 2 × 107 cells/mL. Cells were harvested by centrifugation

at 3000×g for 5 min. Supernatant was removed and the cells were washed with double distilled H2O (ddH2O).

The cells were transferred to one 50 mL falcon tube and washed an additional time with ddH2O before they were

centrifuged at 3000×g for 5 min. Cells were resuspended in 5% glycerol and 10% DMSO to a final concentration of 2 × 109 cells/mL aliquoted in 100 µL. These cells can be

frozen at − 80  °C for several weeks. Before transforma-tion, cells were pelleted and the supernatant removed. For transformation 250 ng of the digested backbone vec-tor and 500  ng of each DNA PCR product (see below)

were added and adjusted to a final volume of 14  µL with ddH2O. The DNA mixture was added to the yeast

along with 260  µL of a 50% (w/v) polyethyleneglycol 3600, 36 µL 1 M lithium acetate and 50 µL of denatured sheared salmon sperm DNA (2 mg/mL). The mixture was vortexed and incubated at 42 °C for 45 min. Cells were centrifuged at 13,000×g for 30 s and the supernatant was removed. The cells were carefully resuspended in 1  mL ddH2O and 200–500  µL were spread on solidified

syn-thetic drop out media containing all necessary supple-ments without uracil for selection. Plates were incubated at 30 °C for 3–5 days.

Plasmid isolation from yeast

All colonies from a transformation plate were scraped and incubated overnight in liquid synthetic yeast drop out solution containing the appropriate supplements without uracil at 200  rpm at 30  °C. One mL was pel-leted and the supernatant removed. 200 µL of STC buffer (50 mM Tris–HCl pH 7.5, 1.2 M sorbitol, 50 mM CaCl2)

including 3  µL Zymolase was added and incubated at 37 °C for 1 h. To the mixture, 200 µL of 1% (w/v) sodium dodecyl sulfate (SDS) in 200 mM NaOH were added and inverted. The solution was neutralized by adding 240 µL of 3 M potassium acetate, pH 5.5 and inverted. The mix-ture was centrifuged and the supernatant mixed with 600  µL isopropanol, inverted and centrifuged at maxi-mum speed for 10  min. The supernatant was removed and the pellet was washed with 70% (v/v) ethanol. The pellet was air dried and resuspended in 30 µL ddH2O.

Transformation of Escherichia coli and plasmid conformation

Following standard techniques [23], 10  µL of the yeast plasmid extract were transformed into E. coli and posi-tive colonies were selected on media containing ampicil-lin. Plasmids from colonies were isolated using standard procedures [23]. Plasmids were restriction digested with appropriate enzymes to confirm correct insertion. Addi-tional confirmation was achieved using PCR amplifica-tion of fused DNA products. To ensure correct DNA sequences for expression plasmids, Sanger sequencing was performed. The correct plasmids were then grown in a 50 mL culture and plasmids were isolated using the Quantum Prep® Plasmid Midiprep Kit (Biorad) accord-ing to the manufacturers’ instructions. Before fungal transformation, the plasmids were linearized using AscI.

Plasmid construction and fungal transformation

(4)

the nitrate inducible aflR/S construct, six fragments total were amplified and eventually cloned into the AscI digested plasmid backbone of pYHC-yA-riboB [57]. The 3′ flanks of stcA and stcW were amplified from A. nidu-lans LO8030 DNA using primer pairs stcA3′-F/-R and stcW3′-F/-R with the -R primers containing 5′ overlaps to the respective site of AscI digested plasmid backbone and the -F primers having overlaps to the aflS termina-tor and pyroA cassette, respectively. The bidirectional niaD/niiA promoter region was amplified from A. nidu-lans LO8030 [15, 38] with overlaps to the open reading frames of aflR and aflS using primer pairs nitrate-F/-R. The open reading frame of aflR including 500 bp of ter-minator was amplified from A. nidulans FGSC 4A DNA using primer pair aflR-F/-R where the -R primer had a 5′ overhang to the terminator region of the A. fumigatus pyroA gene. The open reading frame of aflS was ampli-fied from A. nidulans FGSC 4A DNA using primer pairs aflS-F/-R with the -R primer having a 5′ overlap to the -F primer used to amplify the stcW flank. The pyroA cassette was retrieved through PstI restriction digest of pJMP61 [7]. After yeast recombinational cloning, the plasmid pPMW1 was created. For sterigmatocystin promoter studies the entire bidirectional promoter region between two open reading frames was cloned or, in the case of monodirectional promoters 500 bp upstream of the open reading frame was amplified using primer pairs stc “gene name”-pF/stc “gene name”-pR including 5′ overlaps to the wA 5′ flank and the open reading frame of pyrG gene from A. fumigatus CEA10. Plasmid pYHC-wA-pyrG [55] was linearized using NheI. After yeast recombineering, plasmid pAN “stcGene” were yielded. For constructing the carotenoid expression plasmid pJSF1, the bidirec-tional promoter region between stcA and stcB was ampli-fied using primer pair stcAB-cF/-cR with overlaps to the carotenoid cluster genes carRA and carB from F. fujikuroi IMI58289. The open reading frame of carRA including 500  bp terminator region was amplified from F. fujik-uroi IMI58289 DNA using primer pairs carRA-F/-R with the -R primer including an overlap to the wA 3′ flank of plasmid pYHC-wA-pyrG. The open reading frame of carB including 500  bp terminator region was ampli-fied from F. fujikuroi IMI58289 DNA using primer pairs carB-cF/-cR with the -cR primer including an overlap to the wA 5′ flank of plasmid pYHC-wA-pyrG. All frag-ments were assembled using yeast recombinational clon-ing into EcoRI/XhoI linearized pYHC-wA-pyrG resulting in pJSF1. pJSF2 was assembled in a similar process using EcoRI/XhoI linearized pYHC-yA-riboB and PCR ampli-cons of the stcM promoter (amplified with stcM-cF/-cR) and ggs1 (amplified with ggs1-cF/-cR).

Transformation of A. nidulans was performed as pre-viously described [40]. For selection of nitrate inducible

aflR/S strains, A. nidulans LO8030 was used as the recip-ient strain. pPMW1 was linearized using AscI, trans-formed into LO8030, and transformants were selected on media where pyridoxine was omitted and uracil/uri-dine and riboflavin were supplemented yielding strain TPMW2.3. For selection of stc promoter test strains, TPMW2.3 was used as the recipient strain. pAN “stc-Gene” plasmids were linearized using SbfI and trans-formants were selected on media were riboflavin was omitted and uracil/uridine was supplemented yielding strains TANx and TAASx (see Additional file 1: Table S1). To create a riboflavin prototrophic strain, TPMW2.3 was transformed with SbfI linearized pYHC-yA-riboB and selected on media where riboflavin was omitted and ura-cil/uridine was supplemented yielding strain TPMW7.2. For selection of car expression strains, TPMW7.2 was used as the recipient strain and SbfI linearized pTJSF1 was transformed into TPMW7.2 and transformants selected on media omitting uracil/uridine yielding strain TJSF1.1. An auxotrophic control strain was generated by using TPMW7.2 as recipient strain and SbfI linearized pYHC-wA-pyrG was transformed and selected on media without supplements yielding strain TPMW8.2. For DNA isolation, all fungal strains were grown for 24 h at 37  °C (Aspergillus) or 29  °C (Fusarium) in steady state liquid GMM, supplemented appropriately as described by Shimizu and Keller [48]. Single integration was con-firmed by Southern analysis as described by [23] using P32-labelled probes created by amplification of the

indi-cated DNA fragment in Additional file 2: Figures S1–S4.

Carotenoid analysis

(5)

metabolites was performed using Chromera Manager (PerkinElmer) by comparison to an authentic standard (Sigma Aldrich).

Results

The sterigmatocystin (ST) gene cluster of A. nidulans is known to harbor 25 genes involved in biosynthesis of sterigmatocystin [9]. While environmental regulation of the ST gene cluster is complex and not well understood, it was the first cluster that identified a gene product encoded within the cluster itself to function as a cluster-specific Zn(II)2Cys6 transcription factor, called AflR [21].

The gene encoding AflR shares a bidirectional promoter with aflS encoding a transcriptional cofactor of AflR [20]. We replaced the native promoter of aflR/S with the well characterized niaD/niiA promoter which is induced by the presence of nitrate in the absence of other nitrogen sources [10]. We confirmed nitrate-dependent expression of aflR/S by northern blot analysis (Fig. 1b).

Next, we set out to test which of the 25 stc gene pro-moters would be nitrate-inducible in our production strain (TPMW2.3). Since TPMW2.3 is a uracil/uridine and riboflavin auxotroph, we designed plasmids that contain each of the 25 stc gene promoters, respectively, driving expression of the A. fumigatus pyrG gene along with a riboB selectable marker flanked by bordering regions of the yA locus (Fig. 2a; Additional file 1: Table S3; Additional file 2: Fig. S2). Using a minimalized pro-moter selection strategy, we chose propro-moter regions as follows: For unidirectional stc genes, the promoter region was amplified from the first base after the stop codon of the first gene to the start codon of the second gene, but not exceeding 1  kb. In cases of bidirectional promot-ers, the entire region between the two start codons was chosen, not exceeding 1  kb. We selected 25 strains for each stc promoter for riboflavin prototrophy, exhibit-ing yellow spore color, and a control strain that did not include a stc promoter. To test for the ability of AflR/S to induce AfpyrG expression driven by each of the stc

Fig. 2 Nitrate‑ and stc promoter‑dependent pyrG expression. a Schematic overview of A. niduans stc promoter driven pyrG test strains integrated at the yA locus. bA. nidulans strains were grown for 72 h at 37 °C on solidified GMM plates containing NH4+ or NO3− as nitrogen and half of the plates

(6)

promoters, we grew them on media containing either nitrate (induces aflR/S expression; Fig. 1b) or ammo-nium, and supplemented with or without uracil/uridine (Fig. 2b, c). The growth assay showed that eight of the tested promoters (stcA, stcB, stcI, stcM, stcN, stcQ, stcV, and stcW) exhibited the desired ability to grow on plates without uracil/uridine supplementation on nitrate con-taining media only (Fig. 2b, c), demonstrating specific expression under induction conditions. Three of the pro-moters tested exhibited leaky expression (stcC, stcD, and stcE) as we observed colony growth of strains on media containing ammonium (Fig. 2b, c) where aflR/S should not be induced (Fig. 1b). In order to confirm control of the identified promoters by AflR, we investigated expres-sion of all stc cluster genes in an A. nidulans WT and an isogenic ∆aflR knock-out strain [56] under sterigmato-cystin production conditions. We found that in addition to the eight promoters identified in our plate assay, most of the remaining cluster genes were also expressed in an AflR-dependent manner (Fig. 2d). We speculate that either the length of the chosen promoters, the difference in culture conditions, or the insufficient expression level could be responsible for the observed discrepancies of stc activation of the AfpyrG reporter gene.

To test the functionality of our expression system genes responsible for carotenoid production from Fusar-ium fujikuroi [2–4] were expressed in our A. nidulans nitrate-inducible aflR/S strain TPMW2.3. In F. fujikuroi, the geranylgeranyl diphosphate (GGDP) synthase gene ggs1 is responsible for production of GGDP [34], which is a substrate for CarRA and CarB, encoded by two of the clustered carotenoid biosynthetic genes needed for β-carotene production [31]. We inserted the ggs1 gene driven by the stcM promoter and 0.5  kb of the native terminator, a riboflavin selectable marker flanked by the yA border regions (Additional file 1: Table S3; Addi-tional file 2: Fig. S3), and the two carotenoid cluster genes carRA and carB including 0.5 kb of the native ter-minator regions, responsible for β-carotene production [31] under control of the bidirectional stcA/B promoter flanked by the wA border regions (Additional file 1: Table S3; Additional file 2: Fig. S3). Both plasmids were lin-earized and transformed into TPMW2.3 consecutively, yielding strain TJSF3.1 (Fig. 3a; Additional file 1: Table S1; Additional file 2: Fig. S4). Nitrate-inducible expres-sion of ggs1, carRA, and carB was confirmed by north-ern blot analysis compared to a prototroph control strain that produced white spores (TPMW8.2) (Fig. 3b). When the two strains were grown on nitrate containing media, TJSF3.1 exhibited a characteristic orange color that was absent in the control (Fig. 3c). Characterization of caro-tene production by HPLC showed that the strain TJSF3.1 produced 125  µg β-carotene per gram mycelial dry

weight in our experimental setting (Fig. 3d, e). The pro-duction of β-carotene was significantly higher on nitrate induction media than on non-induction media contain-ing glutamine (Fig. 3d, e). The control strain TPMW8.2 did not show any carotene production (Fig. 3d).

As carotenoid production in N. crassa and Fusarium spp. occurs in both mycelia and spores [5], we asked whether a similar distribution would occur in our pro-duction strain. Therefore, mycelia and spores were assessed individually for β-carotene content. Carot-enoids were only produced in the mycelia and not in the spores (Fig. 4a). Since the GGDP produce by Ggs1 is also utilized for ergosterol production in F. fujikuroi [34] we set out to investigate if the homolog of ggs1 in A. nidulans (AN0654, 54% identity, e-value: 4.0−118)

would be sufficient for β-carotene production. A strain was constructed that only expressed carRA and carB called TJSF1.1 (Additional file 2: Fig. S4). When carote-noid production between TJSF1.1 (carRA and carB) and TJSF3.1 (ggs1, carRA, and carB) was compared no signifi-cant difference under inducing conditions was observed (Fig. 4b), suggesting that AN0654 is sufficient to provide the maximum amount of GGDP that can be funneled into carotenoid production. As it is known that one of the bottlenecks during carotenogenesis is the production of mevalonate (a GGDP precursor) by the 3-hydroxyl-3-me-thyl-glutaryl-conenzyme A reductase (HMG CoA reduc-tase) [1], we grew the two production strains on nitrate media supplemented with mevalonate before carotenoid quantification. However, we did not find any difference in production levels between the strains grown with or without mevalonate (Fig. 4c).

Discussion

(7)

Fig. 3 Nitrate‑dependent β‑carotene production in A. nidulans.a Schematic overview of A. nidulans carotenoid production strain TJSF3.1 that harbors the stcM‑driven F. fujikuroi ggs1 gene at the yA locus and the stcA/B‑driven F. fujikuroi carRA/B genes at the wA locus. b Northern blot expres‑ sion analysis of indicated genes in the indicated strains. The strains were grown in duplicate for 24 h in 50 mL of GMM with 35 mM glutamine (Gln) as nitrogen source for 24 h at 250 rpm at 37 °C. The mycelia were washed and shifted into new media containing either Gln or NO3− as nitrogen

sources and grown at 250 rpm and 37 °C for 1 h before RNA extraction. RNA was visualized as loading control. c Growth on solid medium demon‑ strating carotene expression from strain TJSF3.1 and control strain TPMW8.2 grown for 72 h at 37 °C on solidified GMM media containing nitrate as nitrogen source. Bottom picture shows mycelia of the same strains collected from liquid stationary GMM media containing nitrate as nitrogen source grown for 72 h at 37 °C. d HPLC chromatograms at 453 nm of β‑carotene extracted from indicate strains grown as described in panel (c). e

(8)

The ease of yeast recombinational cloning [39] has been exploited for a wide range of molecular methods, includ-ing gene knock-out libraries [17] and expression sys-tems [47] in filamentous fungi as well as yeast itself [8]. Technically, yeast recombinational cloning allows for the assembly of multiple PCR fragments up to a vector size of several ten thousand kilo bases [39]. One of the major hurdles to overcome during yeast recombineering, is undesired recombination among multiple identical DNA regions. Recently, a study in A. terreus demonstrated the requirement of an AflR-like transcription factor, TerR, for expression of all twelve terrein cluster genes [24] simi-lar to our expression data for AflR-dependency of all 25 stc genes. The system was subsequently used to control one of the terrein promoters in a heterologous expres-sion system in A. niger to demonstrate activation of orsA from A. nidulans [24]. Here, we have demonstrated the specific induction of eight of the 25 stc promoters to control the expression of a reporter gene (pyrG). Subse-quently, we have utilized three of the eight promoters to successfully express three Fusarium spp. derived genes responsible for carotenoid production in A. nidulans and confirm functionality of their gene products by detection of β-carotene.

As a precursor of vitamin A, β-carotene has long been in the focus of biotechnology. The most promi-nent example of heterologous gene expression leading to the production of β-carotene is the development of Golden Rice by Monsanto [37]. However, β-carotenoid production was also achieved in baker’s yeast and bac-teria. The amounts of carotenoids produced by the pro-duction strain constructed in this study are equivalent to the first production strain engineered in yeast [52]. Recent advances in manipulating metabolic pathways and genetic elements have increased the production in

baker’s yeast [8, 30], and similar approaches could be undertaken to increase production in A. nidulans.

Notably, production of β-carotene is restricted to the mycelia in the A. nidulans production strains investi-gated in this study, whereas in other fungal species like Neurospora crassa and F. fujikuroi, carotenes are predom-inantly produced in asexual spores [26, 31]. One explana-tion might be the developmentally controlled expression pattern of the niaD promoter, as it was shown to be only transiently expressed at early stages of conidiation, but not at later time points [33]. Sterigmatocystin and afla-toxin in Aspergillus species are predominantly found in the mycelial fraction and a complex fusion network of vesicles containing different precursors and biosynthetic enzymes that ensure correct cellular localization of these secondary metabolites is being unveiled by several stud-ies [32, 42, 49]. Additionally, pioneering work in A. fumig-atus has demonstrated that certain natural products are predominantly produced in the asexual spores (conidia) [27, 29]. These findings suggest that conidial directed cel-lular pathways in the native host (Fusarium) may differ significantly from Aspergillus as location of β-carotene is not the same in the native and heterologous host. Deter-mining which factors control the direction of fungal natural products to certain developmental structures in different fungal species will be a fascinating future task.

Conclusions

This study presents a new heterologous expression sys-tem for fungal natural products in the genetic model organism A. nidulans. The system described here makes use of the ability to co-express, minimally, eight promot-ers by a fungal-specific Zn(II)2Cys6 transcription factor,

AflR, and its cofactor AflS. By replacing the intrinsic bidirectional aflR/S promoter with a nitrate bidirectional

Fig. 4 Tissue‑ and media‑specific β‑carotene production. a Comparison of β‑carotene production in spores and mycelia of TJSF3.1 in triplicates. The strains were grown under nitrate inducing conditions and carotenoid production was normalized to the amount produced in mycelia. b

Comparison of β‑carotenoid production between TJSF1.1 (carRA and carB) and TJSF3.1 (ggs1, carRA and carB). Strains were grown under nitrate inducible conditions and β‑carotene was quantified based on normalized dry weight in triplicates. No significant difference be could be detected.

(9)

inducible promoter, all eight identified genes can be simultaneously activated and repressed. As all eight iden-tified promoters differ in their DNA sequence, the system has the potential to utilize one-step yeast recombina-tional cloning for assembly of entire secondary metabo-lite gene clusters. Here, we demonstrate the production of β-carotene by heterologous expression of three genes from F. fujikuroi. The inducibility of the system also is useful for production of toxic metabolites at a stage when the host strain has accumulated a significant biomass.

Abbreviations

SM: secondary metabolite; FDA: Food and Drug Administration; ST: sterigmatocystin.

Authors’ contributions

PW, AAS, and NPK conceived and designed the experiments. PW, AAS, JSF, PMW, and AN performed the experiments. NPK provided reagents and materi‑ als. PW, AAS, and NPK wrote the manuscript. All authors read and approved the final manuscript.

Author details

1 Department of Medical Microbiology and Immunology, University of Wis‑ consin, Madison, WI 53706, USA. 2 Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA. 3 Present Address: Hexagon Bio, Menlo Park, CA 94025, USA. 4 Present Address: Department of Cell and Regenerative Biology, University of Wisconsin, Madison, WI 53705, USA. 5 Present Address: Davis Genome Center – Metabolomics, University of California, 451 Health Sci‑ ence Drive, Davis, CA 95616, USA. 6 Present Address: Ocean College, Zhejiang University, Hangzhou 310058, Zhejiang Province, People’s Republic of China.

Acknowledgements

The authors would like to thank Jacob Hagen for excellent technical assistance.

Competing interests

The expression system described in this study has been filed as patent in the United States under Number P150029US01 entitled “Methods and Systems for Producing Fungal Secondary Metabolites” for the Wisconsin Alumni Research Foundation (WARF).

Availability of data and materials

All data generated and analyzed during this study are included in this pub‑ lished article [and its supplementary information files].

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Funding

This work was funded by the Draper Technology Innovation Fund (TIF) from the University of Wisconsin‑Madison to PW and NPK and by funds from AgriMetis, LLC to AAS and NPK. PMW was supported by Grant NSF No. 41406141 from the National Science Foundation. AAS was sponsored by the Genetics Training Program, NIH GM07133, from the University of Wisconsin‑Madison.

Additional files

Additional file 1. Additional tables. Additional file 2. Additional figures.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations.

Received: 21 December 2017 Accepted: 5 March 2018

References

1. Albermann S, Linnemannstöns P, Tudzynski B. Strategies for strain improvement in Fusarium fujikuroi: overexpression and localization of key enzymes of the isoprenoid pathway and their impact on gibberellin biosynthesis. Appl Microbiol Biotechnol. 2013;97:2979–95.

2. Avalos J, Carmen Limón M. Biological roles of fungal carotenoids. Curr Genet. 2015;61:309–24.

3. Avalos J, Estrada AF. Regulation by light in Fusarium. Fungal Genet Biol. 2010;47:930–8.

4. Avalos J, Prado‑Cabrero A, Estrada AF. Neurosporaxanthin production by Neurospora and Fusarium. Methods Mol Biol. 2012;898:263–74. 5. Baima S, Carattoli A, Macino G, Morelli G. Photoinduction of albino‑3

gene expression in Neurospora crassa conidia. J Photochem Photobiol B. 1992;15:233–8.

6. Boecker S, Zobel S, Meyer V, Süssmuth RD. Rational biosynthetic approaches for the production of new‑to‑nature compounds in fungi. Fungal Genet Biol. 2016;89:89–101.

7. Bok JW, Soukup AA, Chadwick E, Chiang YM, Wang CC, Keller NP. VeA and MvlA repression of the cryptic orsellinic acid gene cluster in Aspergillus nidulans involves histone 3 acetylation. Mol Microbiol. 2013;89:963–74. 8. Bond C, Tang Y, Li L. Saccharomyces cerevisiae as a tool for mining, study‑

ing and engineering fungal polyketide synthases. Fungal Genet Biol. 2016;89:52–61.

9. Brown DW, Yu JH, Kelkar HS, Fernandes M, Nesbitt TC, Keller NP, Adams TH, Leonard TJ. Twenty‑five coregulated transcripts define a sterig‑ matocystin gene cluster in Aspergillus nidulans. Proc Natl Acad Sci USA. 1996;93:1418–22.

10. Burger G, Tilburn J, Scazzocchio C. Molecular cloning and functional char‑ acterization of the pathway‑specific regulatory gene nirA, which controls nitrate assimilation in Aspergillus nidulans. Mol Cell Biol. 1991;11:795–802. 11. Butchko RA, Brown DW, Busman M, Tudzynski B, Wiemann P. Lae1

regulates expression of multiple secondary metabolite gene clusters in Fusarium verticillioides. Fungal Genet Biol. 2012;49:602–12.

12. Carberry S, Molloy E, Hammel S, O’Keeffe G, Jones GW, Kavanagh K, Doyle S. Gliotoxin effects on fungal growth: mechanisms and exploitation. Fungal Genet Biol. 2012;49:302–12.

13. Chiang YM, Szewczyk E, Davidson AD, Keller N, Oakley BR, Wang CC. A gene cluster containing two fungal polyketide synthases encodes the biosynthetic pathway for a polyketide, asperfuranone, in Aspergillus nidulans. J Am Chem Soc. 2009;131:2965–70.

14. Chiang YM, Oakley CE, Ahuja M, Entwistle R, Schultz A, Chang SL, Sung CT, Wang CC, Oakley BR. An efficient system for heterologous expression of secondary metabolite genes in Aspergillus nidulans. J Am Chem Soc. 2013;135:7720–31.

15. Chiang YM, Ahuja M, Oakley CE, Entwistle R, Asokan A, Zutz C, Wang CC, Oakley BR. Development of genetic dereplication strains in Aspergillus nidulans results in the discovery of Aspercryptin. Angew Chem Int Ed Engl. 2016;55:1662–5.

16. Clardy J, Walsh C. Lessons from natural molecules. Nature. 2004;432:829–37.

17. Colot HV, Park G, Turner GE, Ringelberg C, Crew CM, Litvinkova L, Weiss RL, Borkovich KA, Dunlap JC. A high‑throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. Proc Natl Acad Sci USA. 2006;103:10352–7.

18. Díaz‑Sánchez V, Estrada AF, Trautmann D, Al‑Babili S, Avalos J. The gene carD encodes the aldehyde dehydrogenase responsible for neurosporax‑ anthin biosynthesis in Fusarium fujikuroi. FEBS J. 2011;278:3164–76. 19. Díaz‑Sánchez V, Limón MC, Schaub P, Al‑Babili S, Avalos J. A RALDH‑like

(10)

We accept pre-submission inquiries

Our selector tool helps you to find the most relevant journal We provide round the clock customer support

Convenient online submission Thorough peer review

Inclusion in PubMed and all major indexing services Maximum visibility for your research

Submit your manuscript at www.biomedcentral.com/submit

Submit your next manuscript to BioMed Central

and we will help you at every step:

20. Ehrlich KC. Predicted roles of the uncharacterized clustered genes in aflatoxin biosynthesis. Toxins (Basel). 2009;1:37–58.

21. Fernandes M, Keller NP, Adams TH. Sequence‑specific binding by Aspergillus nidulans AflR, a C6 zinc cluster protein regulating mycotoxin biosynthesis. Mol Microbiol. 1998;28:1355–65.

22. Fujii I, Ono Y, Tada H, Gomi K, Ebizuka Y, Sankawa U. Cloning of the pol‑ yketide synthase gene atX from Aspergillus terreus and its identification as the 6‑methylsalicylic acid synthase gene by heterologous expression. Mol Gen Genet. 1996;253:1–10.

23. Green MR, Sambrook J. Molecular cloning: a laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 2012.

24. Gressler M, Hortschansky P, Geib E, Brock M. A new high‑performance heterologous fungal expression system based on regulatory ele‑ ments from the Aspergillus terreus terrein gene cluster. Front Microbiol. 2015;6:184.

25. Kealey JT, Liu L, Santi DV, Betlach MC, Barr PJ. Production of a polyketide natural product in nonpolyketide‑producing prokaryotic and eukaryotic hosts. Proc Natl Acad Sci USA. 1998;95:505–9.

26. Li C, Schmidhauser TJ. Developmental and photoregulation of al1 and al2, structural genes for two enzymes essential for carotenoid biosynthe‑ sis in Neurospora. Dev Biol. 1995;169:90–5.

27. Lim FY, Keller NP. Spatial and temporal control of fungal natural product synthesis. Nat Prod Rep. 2014;31:1277–86.

28. Lim FY, Hou Y, Chen Y, Oh JH, Lee I, Bugni TS, Keller NP. Genome‑based cluster deletion reveals an endocrocin biosynthetic pathway in Aspergillus fumigatus. Appl Environ Microbiol. 2012;78:4117–25.

29. Lim FY, Ames B, Walsh CT, Keller NP. Co‑ordination between BrlA regula‑ tion and secretion of the oxidoreductase FmqD directs selective accu‑ mulation of fumiquinazoline C to conidial tissues in Aspergillus fumigatus. Cell Microbiol. 2014;16:1267–83.

30. Lin YJ, Chang JJ, Lin HY, Thia C, Kao YY, Huang CC, Li WH. Metabolic engineering a yeast to produce astaxanthin. Bioresour Technol. 2017;245:899–905.

31. Linnemannstöns P, Prado MM, Fernández‑Martín R, Tudzynski B, Avalos J. A carotenoid biosynthesis gene cluster in Fusarium fujikuroi: the genes carB and carRA. Mol Genet Genomics. 2002;267:593–602.

32. Linz JE, Chanda A, Hong SY, Whitten DA, Wilkerson C, Roze LV. Proteomic and biochemical evidence support a role for transport vesicles and endosomes in stress response and secondary metabolism in Aspergillus parasiticus. J Proteome Res. 2012;11:767–75.

33. Marcos AT, Ramos MS, Marcos JF, Carmona L, Strauss J, Cánovas D. Nitric oxide synthesis by nitrate reductase is regulated during development in Aspergillus. Mol Microbiol. 2016;99:15–33.

34. Mende K, Homann V, Tudzynski B. The geranylgeranyl diphosphate synthase gene of Gibberella fujikuroi: isolation and expression. Mol Gen Genet. 1997;255:96–105.

35. Meyer V, Nevoigt E, Wiemann P. The art of design. Fungal Genet Biol. 2016;89:1–2.

36. Newman DJ, Cragg GM. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod. 2012;75:311–35.

37. Normile D. Agricultural biotechnology. Monsanto donates its share of golden rice. Science. 2000;289:843–5.

38. Oakley CE, Ahuja M, Sun WW, Entwistle R, Akashi T, Yaegashi J, Guo CJ, Cerqueira GC, Russo Wortman J, Wang CC, Chiang YM, Oakley BR. Discov‑ ery of McrA, a master regulator of Aspergillus secondary metabolism. Mol Microbiol. 2017;103:347–65.

39. Oldenburg KR, Vo KT, Michaelis S, Paddon C. Recombination‑mediated PCR‑directed plasmid construction in vivo in yeast. Nucleic Acids Res. 1997;25:451–2.

40. Palmer JM, Perrin RM, Dagenais TR, Keller NP. H3K9 methylation regu‑ lates growth and development in Aspergillus fumigatus. Eukaryot Cell. 2008;7:2052–60.

41. Peláez F. The historical delivery of antibiotics from microbial natural products—can history repeat. Biochem Pharmacol. 2006;71:981–90. 42. Pfannenstiel BT, Zhao X, Wortman J, Wiemann P, Throckmorton K, Spraker

JE, Soukup AA, Luo X, Lindner DL, Lim FY, Knox BP, Haas B, Fischer GJ, Choera T, Butchko RAE, Bok JW, Affeldt KJ, Keller NP, Palmer JM. Revitaliza‑ tion of a forward genetic screen identifies three new regulators of fungal secondary metabolism in the genus Aspergillus. MBio. 2017;8:e01246‑17.

43. Porquier A, Morgant G, Moraga J, Dalmais B, Luyten I, Simon A, Pradier JM, Amselem J, Collado IG, Viaud M. The botrydial biosynthetic gene cluster of Botrytis cinerea displays a bipartite genomic structure and is positively regulated by the putative Zn(II)2Cys6 transcription factor BcBot6. Fungal Genet Biol. 2016;96:33–46.

44. Punt PJ, Greaves PA, Kuyvenhoven A, van Deutekom JC, Kinghorn JR, Pouwels PH, van den Hondel CA. A twin‑reporter vector for simultane‑ ous analysis of expression signals of divergently transcribed, contiguous genes in filamentous fungi. Gene. 1991;104:119–22.

45. Punt PJ, Strauss J, Smit R, Kinghorn JR, van den Hondel CA, Scazzocchio C. The intergenic region between the divergently transcribed niiA and niaD genes of Aspergillus nidulans contains multiple NirA binding sites which act bidirectionally. Mol Cell Biol. 1995;15:5688–99.

46. Qiao J, Kontoyiannis DP, Wan Z, Li R, Liu W. Antifungal activity of statins against Aspergillus species. Med Mycol. 2007;45:589–93.

47. Schumacher J. Tools for Botrytis cinerea: New expression vectors make the gray mold fungus more accessible to cell biology approaches. Fungal Genet Biol. 2012;49:483–97.

48. Shimizu K, Keller NP. Genetic involvement of a cAMP‑dependent protein kinase in a G protein signaling pathway regulating morphological and chemical transitions in Aspergillus nidulans. Genetics. 2001;157:591–600. 49. Soukup AA, Fischer GJ, Luo J, Keller NP. The Aspergillus nidulans Pbp1

homolog is required for normal sexual development and secondary metabolism. Fungal Genet Biol. 2017;100:13–21.

50. Sun WW, Guo CJ, Wang CCC. Characterization of the product of a non‑ ribosomal peptide synthetase‑like (NRPS‑like) gene using the doxycy‑ cline dependent Tet‑on system in Aspergillus terreus. Fungal Genet Biol. 2016;89:84–8.

51. Wiemann P, Keller NP. Strategies for mining fungal natural products. J Ind Microbiol Biotechnol. 2013;41:301–13.

52. Yamano S, Ishii T, Nakagawa M, Ikenaga H, Misawa N. Metabolic engineer‑ ing for production of beta‑carotene and lycopene in Saccharomyces cerevisiae. Biosci Biotechnol Biochem. 1994;58:1112–4.

53. Yeh HH, Ahuja M, Chiang YM, Oakley CE, Moore S, Yoon O, Hajovsky H, Bok JW, Keller NP, Wang CC, Oakley BR. Resistance gene‑guided genome mining: Serial promoter exchanges in Aspergillus nidulans reveal the bio‑ synthetic pathway for fellutamide B, a proteasome inhibitor. ACS Chem Biol. 2016;11:2275–84.

54. Yin WB, Baccile JA, Bok JW, Chen Y, Keller NP, Schroeder FC. A nonriboso‑ mal peptide synthetase‑derived iron(III) complex from the pathogenic fungus Aspergillus fumigatus. J Am Chem Soc. 2013;135:2064–7. 55. Yin WB, Chooi YH, Smith AR, Cacho RA, Hu Y, White TC, Tang Y. Discovery

of cryptic polyketide metabolites from dermatophytes using heterolo‑ gous expression in Aspergillus nidulans. ACS Synth Biol. 2013;29:549–55. 56. Yu JH, Butchko RA, Fernandes M, Keller NP, Leonard TJ, Adams TH. Con‑

servation of structure and function of the aflatoxin regulatory gene aflR from Aspergillus nidulans and A. flavus. Curr Genet. 1996;29:549–55. 57. Zhang P, Wang X, Fan A, Zheng Y, Liu X, Wang S, Zou H, Oakley BR, Keller

Figure

Fig. 1 Nitrate‑inducible inducible aflR/S expression in A. nidulans. a Schematic overview of the aflR/S expression strain TPMW2.3 that harbors the nitrate‑niaD/niiA promoter‑driven aflR/S genes at the native sterigmatocystin cluster locus
Fig. 2 Nitrate‑ and were supplemented with 5 mM uracil/uridine as indicated. Green boxes indicate promoters with desirable traits of strict nitrate inducibility
Fig. 3 Nitrate‑dependent β‑carotene production in Quantification of β‑carotene produced by TJSF3.1 grown on either  NOsources and grown at 250 rpm and 37 °C for 1 h before RNA extraction
Fig. 4 Tissue‑ and media‑specific β‑carotene production. cinducible conditions and β‑carotene was quantified based on normalized dry weight in triplicates

References

Related documents

The aim of the present study was to assess the relationship between cervical length measurement by transvaginal ultrasonography and time to delivery within 7 days in term

Our results about association of residents’ perceptions for benefits and costs of sustainable tourism development and their support for sustainable tourism

Conclusion: Among the analyzed factors weight <2500gm, gestation <34weeks, initial arterial pH <7.1, O 2 saturation<80%, PaCO 2 >60mmHg, FiO 2 >60%,

The effect of human capital and other variables such as education (length time in school), participation in certified job training, length of employment in the

In this paper, we propose an MCS task allocation frame- work to protect participants’ location privacy with differen- tial geo-obfuscation, while minimizing the selected

It modifies the degree argument provided by the covert degree head and constrains the relevant difference degrees to small degrees thus making the difference between the